Markakis became the first two-time winner of that award in 2003 when he had "one of the greatest two-way seasons in junior college history" according to Baseball America. He went 12-0 with a 1.68 ERA and hit .439 with 21 homers. At the plate, he led the nation in RBI (92) and on the mound, he led in strikeouts (160). He turned a $1.5 million offer from the Reds and re-entered the draft. The Baltimore Orioles took him with the 7th overall pick in the 2003 amateur draft and he was signed by scout Dave Jennings for a $1,850,000 bonus. In the 2003 European Championship, Markakis batted .323/~.364/.355 but led the Greek national team with 7 RBI in 8 games to help them finish in second place. He also pitched one scoreless inning.

In 2005, Markakis hit .300/.379/.480 with the Frederick Keys, earning an August call-up to the Bowie BaySox, where he batted .339/.420/.573. He hit 41 doubles that year. Continuing to earn high praise from Baseball America, he was picked as the Orioles Minor League Player of the Year, the #6 prospect in the Eastern League, the top batting prospect, power prospect and strike-zone judgement hitter in the Carolina League and the #2 prospect in that loop, between Jarrod Saltalamacchia and Anibal Sanchez. He made the Carolina League All-Star team and was 7th in the league in average.

Nick became the regular 2006 Orioles right fielder and batted .291/.351/.448 for the season. However, he started very slowly, hitting only .182 in April, and while he improved each month, he was still hitting only .257 at the end of June. Hitting over .400 in July and over .350 in August helped a lot to raise his average. He played 161 games in 2007, hitting 43 doubles,d 23 homers and 112 RBI as part of a batting line of .300/.362/.485, making him an outstanding offensive performer, although just a notch below All-Star level. Even though he was only 23 at the time, he never took that last step to stardom, but instead settled at that level, collecting an OPS+ above 100 year after year and standing out as the most dependable player on a perennially week Orioles team.

In 2008, Markakis had his best season, hitting .306 with 48 doubles and 20 homers in 157 games. His 106 runs scored were a career high, as was his OPS+ of 136. In 2009, he fell back slightly, to .293/.347/.453 in 161 games. He collected 101 RBI that season, then in 2010 hit .297 in 160 games. He continued with his large number of doubles, with 45, his fourth-straight season over 40, but his home run production fell to only 12. As a result, and with the Orioles struggling even more than usual,he scored only 79 runs and drove in a mere 60. In 2011 however, he fell to .284 in 160 games, with 31 doubles and 15 homers, the lowest number of extra-base hits in his career. However, he won a Gold Glove as one of the American League's best defensive outfielders, and his OPS+ was still well positive at 107.

In 2012, the Orioles suddenly became good in a completely unexpected development. Markakis was his usual self, hardly missing a game, hitting just below .300, while producing his share of extra-base hits and drawing a large number of walks. In mid-season, he was moved to the lead-off spot in the line-up, after injuries to Brian Roberts and Nolan Reimold, and he did very well, hitting .336 in the role. He was hitting .297/.361/.471 in 103 games with 13 homers in 103 games when his thumb was fractured by a CC Sabathia pitch in the 5th inning of a key game against the New York Yankees on September 8th. He had already missed the month of June with a broken hamate bone in his right hand - the first time he had missed any playing time since coming up in 2006 - and the second fracture put him out for the rest of the season with his team tied with the Yankees with three weeks left to play. The Orioles did make the postseason, but he was unable to play in any of the games. In 2013, he was back in full health, playing 160 games with a .271 average, 10 homers and 59 RBIOs, although he did score 89 runs. Still, the season was well below par for him, as for the first time his OPS+ fell below 100, to 88, the result of the drop in both power and batting average. He bounced back in 2014 however, as did the Orioles, who ran away with the AL East title with a strong second half. As the team's lead-off hitter he hit .276 with 14 homers and 50 RBIs and scored 81 runs, his OPS+ moving back to his customary range at 106. For the first time he was able to play in the postseason, going a combine 8 for 31 (.258) with a homer an 3 RBIs in two rounds of playoff action. He ended his Orioles career with 1365 games played for the team.

On December 3, 2014, Markakis signed a four-year free agent contract with the Atlanta Braves worth a total of $44 million. The Braves had traded starting RF [[Jason Heyward] earlier that off-season and were looking to Markakis to replace him in 2015. Shortly after signing the contract, he underwent surgery on a herniated disk in his neck. As he arrived in spring training, he explained that the neck surgery was the main reason he did not re-sign with the Orioles, because GM Dan Duquette did not want to take the risk that it would affect his production down the line.